Methods of this type are widely known.
The specific detection of biomolecules, more particularly of proteins and/or peptides from small amounts of biological material is an important requirement for research and medical diagnostics.
Therefore, the aim of new developments is to increase the sensitivity of known detection methods, more particularly of protein detection methods. More particularly, the aim is to be able to detect and quantify a larger number of different proteins or protein variants at the same time in one sample.
Technological developments which have brought significant advances in the detection of proteins are in particular mass spectrometry and microarray based approaches.
Mass spectrometric methods are capable of detecting a large number of different proteins in an unambiguous manner from a complex sample. Sensitivity is a critical issue with that technology, despite major advances in separation and detection techniques, since the sensitive detection of proteins from small sample amounts is frequently not possible due to system limitations.
A second critical issue is the cost associated with mass spectrometric analysis. To allow specific protein detection, complicated and cost-intensive sample preparation and protein separation techniques are required. Thus, the costs for the equipment required for this purpose, i.e., the actual mass spectrometer, are very high.
The result has been that mass spectrometric analyses are carried out in a few specialized laboratories and only a small portion of the diagnostic detection methods available are based on this technique.
Immunoassays such as immunohistochemistry, direct immunoassay and sandwich immunoassay are, therefore, vastly more important analytical methods in research laboratories and also in clinical analyses. They generally allow the specific detection of proteins in complex samples without complicated sample preparation.
The achievable sensitivity can reach as low as the femtomolar concentration range, and specificity of the measurement signal can be very high.
In recent years, great efforts have been made to bring the immunoassay from the determination of individual parameters into a multiplex format so that it is possible to detect a larger number of different proteins at the same time.
For example, protein microarrays, which in the form of solid-phase assays on planar supports or on microspheres represent a significant miniaturization of immunoassays, have been developed which allow detection and quantification of dozens to hundreds of different proteins from a few microliters of sample volume.
In those antibody-based approaches, a first problem is the lack of binding affinity molecules, i.e., of antibodies, which are a prerequisite for a sensitive and specific immunoassay. A second problem is cross-reactivity of the antibodies used with one another and cross-reactivity with different analyte proteins.
Those limitations frequently result in the sensitivity of the detection method being distinctly lowered when more than about 20 proteins are simultaneously detected.
Immunoblots, also known as Western blots, are also known, which first comprise using gel electrophoresis in a support matrix to separate proteins according to their size, charge or other physical properties into bands and then transferring the bands onto a membrane, where the proteins are then accessible for antibody binding events.
That method is hampered by the same disadvantages as the above-described immunoassays.
In view of the above, it could be helpful to provide a simple and inexpensively implementable method of the type mentioned at the outset, with which it is possible to specifically and sensitively detect from small amounts of biological material a multiplicity of biomolecules, more particularly, proteins, peptides or proteins digested to form peptides.